Processes for preparing oxathiazin-like compounds

ABSTRACT

Oxathiazin-like compounds, processes for making new oxathiazin-like compounds, compounds useful for making oxathiazin-like compounds, and their uses are disclosed. Processes of treating patients suffering from cancers, bacterial infections, fungal infections and/or viral infections by administering oxathiazin-like compounds are also disclosed. These compounds were found to have significantly longer half-life compared to taurolidine and taurultam.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to new compounds, processes for preparingnew compounds and uses thereof.

Description of the Background Art

Oxathiazin-like compounds are known from U.S. Pat. No. 3,202,657 andU.S. Pat. No. 3,394,109.

There remains a need in the art for new compounds and processes formaking such compounds to provide compounds with more potentantineoplastic and antimicrobial activity, less toxicity and sideeffects, and less resistance to treatment by tumor or microbial cells.

SUMMARY OF THE INVENTION

In accordance with the present invention, new oxathiazin-like compounds,processes for making new oxathiazin-like compounds, compounds useful formaking oxathiazin-like compounds, and their uses are disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically shows anti-neoplastic activity of one embodiment ofthe invention in a cytotoxicity assay in LN-229 cells.

FIG. 2 graphically shows anti-neoplastic activity of one embodiment ofthe invention in a cytotoxicity assay in SW480 (human colonadenocarcinoma) cells.

FIG. 3A-3C Cytotoxicity induced in murine SMA 560 bulk glioma cellsafter treatment with taurolidine and taurultam (TT). Cytotoxicity wasassessed after 24 h (FIG. 3A) and 48 h (FIG. 3B) of treatment. The EC₅₀values for taurolidine (34.6 μg/ml) and taurultam (19.3 μg/ml) are givenin the lower panel (FIG. 3C). Data are presented as mean values±SD ofthree independent experiments.

FIG. 4 Cytotoxicity induced by taurolidine and taurultam (TT) in murineSMA560 glioma cancer stem cells (CSC). Data are presented as meanvalues±SD.

FIG. 5A-5C Cytotoxicity induced in cancer stem cells isolated from fourglioblastoma multiforme (GBM) patients (GBM #3, #4, #5 and #6) aftertreatment for 24 h with taurolidine (FIG. 5A), taurultam (TT) (FIG. 5B)or temozolamide (FIG. 5C). Data are presented as mean values±SD.

FIG. 6 FTIR spectrum of compound 2244 made according to the presentinvention.

FIG. 7 FTIR spectrum of compound 2250 made according to the presentinvention.

FIG. 8 shows the results of a spheroid toxicity assay for multicellularpancreatic tumor (Panc Tul or BxPC-3) spheroids in which control,taurolidine-treated (500 μM) or compound 2250-treated (1000 μM) sampleswere treated for 48 hours (columns labeled A) and strained to testresidual aggregates (columns labeled B) for stability.

FIGS. 9A and 9B show the results of FACS analysis of the Panc Tulmulticellular spheroid cultures CD133 content.

FIG. 10A shows MiaPaca2 tumor volume upon treatment with control ortaurolidine. FIG. 10B shows MiaPaca2 tumor volume upon treatment withcontrol or compound 2250. FIG. 10C shows PancTu I tumor volume upontreatment with control or taurolidine. FIG. 10D shows PancTu I tumorvolume upon treatment with control or compound 2250.

FIG. 11A is a xenograft model of pancreatic primary tumors (Bo 73)observed for 15 days when treated with control, taurolidine or compound2250. FIG. 11B is a xenograft model of pancreatic primary tumors (Bo 70)observed for 23 days when treated with control, taurolidine or compound2250.

DETAILED DESCRIPTION OF THE INVENTION

According to certain embodiments, the present invention relates tooxathiazin-like compounds, as well as derivatives thereof and processesand compounds for preparing oxathiazin-like compounds and derivativesthereof.

Oxathiazin-like compounds and derivatives thereof according to certainembodiments of the present invention have antineoplastic activities,antimicrobial activities and/or other activities.

Processes for making oxathiazin-like compounds and derivatives thereofaccording to certain embodiments of this invention provide advantageousmethods for making compounds having antineoplastic activities,antimicrobial activities and/or other activities. In certainembodiments, oxathiazin-like compounds and derivatives thereof areuseful, inter alia, in the treatment of cancers and tumors in a subject,such as a human patient. Accordingly, in certain embodiments the presentinvention also relates to treatment of cancers and tumors usingcompounds described herein. Cancers such as central nervous systemcancers including glioblastoma, glioma, neuroblastoma, astrocytoma, andcarcinomatous meningitis, colon cancer, rectal cancer and colo-rectalcancer, ovarian cancer, breast cancer, prostate cancer, lung cancer,mesothelioma, melanoma, renal cancer, liver cancer, pancreatic cancer,gastric cancer, esophageal cancer, urinary bladder cancer, cervicalcancer, cardiac cancer, gall bladder cancer, skin cancer, bone cancer,cancers of the head and neck, leukemia, lymphoma, lymphosarcoma,adenocarcinoma, fibrosarcoma, and metastases thereof, for example, arediseases contemplated for treatment according to certain embodiments ofthe invention. Drug resistant tumors, for example a multiple drugresistant (MDR) tumor, also are useful in certain embodiments using theinventive compounds, including drug resistant tumors which are solidtumors, non-solid tumors and lymphomas. It is presently believed thatany neoplastic cell can be treated using the methods described herein.

Tumor stem cells (also referred to as cancer stem cells (CSCs)) areconsidered to be the main drivers for the formation of metastases andthe regrowth of tumors after resection.

In certain embodiments, compounds of the present invention are useful,inter alia, in the treatment of tumor stem cells in a subject.

In certain embodiments, compounds of the present invention are useful,inter alia, in the treatment of glioblastoma tumor stem cells in asubject.

In certain embodiments, the invention kills tumor cells and/or CSCs, orinhibits their growth, by oxidative stress, apoptosis and/or inhibitinggrowth of new blood vessels at the tumor site (anti-angiogenesis andanti-tubulogenesis). A primary mechanism of action for killing tumorcells and/or CSCs is oxidative stress. Tumor cells and/or CSCs may alsobe killed by apoptosis according to the invention. At lower bloodconcentrations, compounds according to the invention are effective atinhibiting tumor cell growth by their anti-angiogenic action and theiranti-tubulogenic action, and these compounds are thus useful forpalliative treatment.

Oxathiazin-like compounds and derivatives thereof of the inventionmetabolize much slower in the bloodstream than taurolidine andtaurultam. Accordingly, lower doses of such compounds can beadministered to a patient to achieve similar effects.

It was unexpectedly found that within minutes of exposure totaurolidine, tumor cells react by initiating the program of apoptoticcell death as follows:

-   -   1. The primary insult of Taurolidine to the tumor cell is an        increase of reactive oxygen species (ROS), which is measured        fluorimetrically.    -   2. The induction of oxidative stress by Taurolidine as the        primary step is supported by the finding that the antineoplastic        action of Taurolidine can be prevented by the addition of a        reducing agent such as glutathione or N-acetylcysteine.    -   3. The damage caused by the elevated ROS to the mitochondria of        the tumor cell results in the loss of their membrane potential        and the release of Apoptosis Inducing Factor (Al F).    -   4. AIF is translocated to the nucleus and initiates the        expression of pro-apoptotic genes, which results in the blebbing        of the plasma membrane, in chromatin condensation and DNA        fragmentation, the hallmarks of apoptosis.    -   5. In contrast to normal cells, tumor cells are very sensitive        to oxidative stress. This explains the action of Taurolidine        against a broad range of tumor cells, sparing normal cells.

Compounds of the present invention also are useful, in certainembodiments, in treatment of microbial infections in a subject, such asa human patient. Microbial infections which may be treated accordingcertain embodiments include bacterial infections, fungal infectionsand/or viral infections.

Cancer patients tend to be immunocompromised, making them particularlysusceptible to microbial infections, especially during and/or aftersurgery.

In certain embodiments, compounds of the invention are utilized to treatglioblastoma in a subject.

In certain embodiments, compounds of the invention are utilized to treatS. aureus infection in a subject.

In certain embodiments, compounds of the invention are utilizedaccording to the invention to treat MRSA in a subject.

In certain embodiments, compounds of the invention are utilizedaccording to the invention to treat E. coli in a subject.

In certain embodiments, compounds of the invention are utilizedaccording to the invention to treat H. pylori in a subject, and/orcancer(s) associated with H. pylori in a subject.

In certain embodiments, compounds of the invention are utilizedaccording to the invention to treat HIV in a subject.

In certain embodiments, compounds according to formula I are utilizedaccording to the invention wherein R is H, alkyl, or the like, such asmethyl, ethyl, propyl, (e.g., isopropyl), benzyl or the like.

In certain embodiments, new compound 2250(Tetrahydro1,4,5-oxathiazin-4-dioxide or 1,4,5-oxathiazan-4-dioxide) isprepared and/or utilized according to the invention. An FTIR spectrumfor compound 2250 made according to the present invention is shown inFIG. 8.

In certain embodiments, new compound 2245 is prepared and/or utilizedaccording to the invention.

Compound 2250 prevents and treats stomach tumors, including tumorscaused by or associated with H. pylori, or tumors as a consequence ofmetastasis to the stomach.

The amount of the compounds needed depends on tumor size. In oneembodiment, the invention includes surgically reducing tumor size andtreating with one or more of the compounds. The compound may beadministered before, during or after surgery to reduce tumors. Compoundsaccording to the invention can be administered by any suitable method,including without limitation, by gels, capsules, tablets, IV, IP and/ordirectly to the tumor.

Gels can contain for example 2-4% (e.g., 3%) active compound of theinvention, such as compound 2250, alone or in combination withtaurolidine/taurultam which also can be administered and present alone,and can be for topical administration. Such gels can be used to treattumors of the skin and mouth, including squamous cell tumors of themouth and skin. Such gels also can be used to treat cervical cancer orcervical dysplasia by being administered in a suppository to the vagina,or by syringe. The invention may include the combination of asuppository carrying an active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the providedcomposition is mixed with at least one inert, pharmaceuticallyacceptable excipient and/or fillers or extenders (e.g., starches,lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g.,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia), humectants (e.g., glycerol), disintegrating agents(e.g., agar, calcium carbonate, potato starch, tapioca starch, alginicacid, certain silicates, and sodium carbonate), solution retardingagents (e.g., paraffin), absorption accelerators (e.g., quaternaryammonium compounds), wetting agents (e.g., cetyl alcohol and glycerolmonostearate), absorbents (e.g., kaolin and bentonite clay), andlubricants (e.g., talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate), and mixtures thereof. Inthe case of capsules, tablets and pills, the dosage form may comprisebuffering agents.

The compounds of this disclosure, particularly compound 2250, have beenfound to be very soluble in water. In certain embodiments, no PVPnecessary to increase the solubility. For example, a 3.2% solution 2250is isotonic. This is an unexpected advantage over taurolidine.

Compounds of the invention, such as compound 2250 (with or withouttaurolidine and/or taurultam) are particularly useful in surgicaloncology, since the compounds do not hinder wound healing.Administration of other antineoplastic drugs must be delayed for up tofive weeks or more after surgery because other such antineoplastic drugshinder wound healing and promote anastomotic leakage. Such problems canbe avoided with compounds of the invention such as compound 2250, whichcan be administered during surgery and immediately thereafter, withoutwound healing issues or leakage issues.

Solid compositions of a similar type may be employed as fillers in softand/or hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the provided composition(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

In certain embodiments, capsules may contain an excipient formulationcontaining one or more of hydroxypropyl methylcellulose (HPMC), gelatin,and fish gelatin. In certain embodiments, a capsule may contain compound2250 in combination with taurolidine and/or taurultam. The capsule mayoptionally further contain one or more of lycopene, ellagic acid(polyphenol), curcumin, piperine, delphinidin, resveratrol,isothiocyanates such as sulforaphane, capsaicin, and piperlongumine.

Active compounds of the invention, such as compound 2250, can becombined with compounds such as gemcitabine. This combination can beused to treat cancers, such as pancreatic cancer. Taurolidine and/ortaurultam also can be combined with gemcitabine to treat, for example,pancreatic cancer.

In some embodiments, a nutritional cancer prophylaxis and treatmentproduct may contain 100-500 mg compound 2250 alone or in combinationwith 100-500 mg taurolidine and/or taurultam and one or more oflycopene, e.g., 20-200 mg, ellagic acid (polyphenol), curcumin, piperine(20-200 mg), delphinidin, resveratrol, isothiocyanates such assulforaphane, capsaicin, and piperlongumine.

It was unexpectedly found that the compounds could be administeredduring surgery and immediately after surgery because the compounds donot inhibit wound healing like other chemotherapy agents.

It was unexpectedly found that taurolidine, taurultam, andoxathiazin-like compounds and derivatives thereof kill tumor stem cells,which is very unusual and perhaps unknown among chemotherapy agents.Typical chemotherapy agents, if effective against tumor stem cells,generally are only effective at very high doses which are extremelytoxic to human patients.

It was unexpectedly found that lower doses of taurolidine and/ortaurultam killed tumor stem cells than were needed to kill tumor cells.

It was unexpectedly found that Oxathiazin-like compounds and derivativesthereof have a half-life in human blood that is significantly longerthan the half-life of taurolidine and taurultam. Accordingly, thesecompounds are cleared less rapidly from the bloodstream of the patients,thereby effectively delaying loss of drug potency caused by the body'sclearance mechanisms.

It was unexpectedly found that certain Oxathiazin-like compounds andderivatives thereof have reduced burning sensation when applied directlyinto tissue, unlike this effect observed in patients treated withtaurolidine.

It was unexpectedly found that the Oxathiazin-like compounds andderivatives thereof have a particularly advantageous combination ofproperties including high water solubility, versatile administrationroutes including oral and i.v., extended stability and half-life, andreduced side effect of burning sensation.

Thus, the half-life of compound 2250 is greater than 24 hours in humanblood, which is significantly higher than the half-life of taurolidine,which was found to be ˜30 minutes using the same test.

In one embodiment, the invention includes treating a patient byadministering compound 2250 to the patient that results in a baselineblood concentration of compound 2250 within about 5 minutes ofadministration. The method involves maintaining a blood concentration ofcompound 2250 in the patient that is about 80% of the baseline bloodconcentration for about 20 hours.

In one embodiment, the invention includes maintaining a bloodconcentration of an anti-neoplastic compound in a patient that is about80% of the patient's baseline blood concentration for about 20 hours byadministering a daily dosage of compound 2250 once daily to maintain theblood concentration that is 80% of the baseline blood concentration.

The daily dosage may be about 0.1 g to about 100 g, e.g., about 5 g toabout 30 g. The daily dosage may be administered in the form of anorally administrable composition. The daily dosage may be administeredin the form of a capsule, a tablet, or a pharmaceutically acceptablesolution. The daily dosage may be administered in a form that containscompound 2250 at a concentration of about 0.01 to about 3% w/v. Thedaily dosage may be administered in a form that contains compound 2250at a concentration of about 0.01 μg/ml to about 1000 μg/ml. The dailydosage may be administered in a form that contains one or moresolubilizing agents, e.g., polyols.

In some embodiments, the compounds are administered in compositions at aconcentration of about 0.01 to about 1000 μg/ml. In some embodiments,the compounds are administered in compositions at a concentration ofabout 1 to about 100 μg/ml. In some embodiments, the compounds areadministered in compositions at a concentration of about 10 to about 50μg g/ml. The composition may also contain about 0.01 to about 1000μg/ml, about 1 to about 100 μg/ml, or about 10 to about 50 μg g/mltaurolidine and/or taurultam.

In some embodiments, the compounds are administered in compositions at aconcentration of about 0.01 to about 3%. In some embodiments, thecompounds are administered in compositions at a concentration of about0.1 to about 2.5%. In some embodiments, the compounds are administeredin compositions at a concentration of about 1% to about 2%. Thecomposition may additionally contain about 0.01 to about 3%, about 0.1to about 2.5%, or about 1 to about 2% taurolidine and/or taurultam.

In one embodiment, the oxathiazin-like compounds and derivatives thereofmay be administered as a co-therapy with taurolidine and/or taurultam tokill tumor stem cells. In accordance with such an embodiment, theco-therapy has been unexpectedly found to require a lower dosage of drugto kill tumor stem cells than necessary to kill normal tumor cells.

In certain embodiments, the oxathiazin-like compounds and derivativesthereof may be administered with Vitamin D3, which results to increasethe anti-tumor effects of the compounds.

In one embodiment, the compound is administered to the subject at atotal daily dose of from about 0.1 g to about 100 g, about 1 g to about80 g, about 2 g to about 50 g, or about 5 g to about 30 g.

Effective dosage amounts of the compounds are dosage units within therange of about 0.1-1,000 mg/kg, preferably 150-450 mg/kg per day, andmost preferably 300-450 mg/kg per day.

As used herein, the term pure refers to a substance that is at leastabout 80% pure of impurities and contaminants. In some embodiments, theterm pure refers to a substance that is at least about 90% pure ofimpurities and contaminants. In certain embodiments, the term purerefers to a substance that is at least about 95% pure of impurities andcontaminants. In some embodiments, the term pure refers to a substancethat is at least about 99% pure of impurities and contaminants. In someembodiments, the term pure refers to a substance that is at least about99.5% pure of impurities and contaminants.

In certain embodiments, compounds, compositions, and methods of thepresent invention encompass the use of micronized compounds. In someembodiments, the term “micronized” as used herein refers to a particlesize in the range of about 0.005 to 100 microns. In certain embodiments,the term “micronized” as used herein refers to a particle size in therange of about 0.5 to 50 microns. In certain embodiments, the term“micronized” as used herein refers to a particle size in the range ofabout 1 to 25 microns. For example, the size of the drug particles maybe about 1, 5, 10, 15, 20, or 25 microns.

In certain embodiments, compounds, compositions, and methods of thepresent invention encompass the use of nanoparticles. As used herein,the term “nanoparticle” refers to any particle having a diameter of lessthan 1000 nanometers (nm). In some embodiments, a nanoparticle has adiameter of less than 300 nm. In some embodiments, a nanoparticle has adiameter of less than 100 nm. In some embodiments, a nanoparticle has adiameter of less than 50 nm, e.g., between about 1 nm and 50 nm.Suitable formulations for injection or infusion may comprise an isotonicsolution containing one or more solubilizing agents, e.g., polyols suchas glucose, in order to provide solutions of increased compoundconcentration. Such solutions are described in EP 253662B1. The solutioncan be rendered isotonic with ringer solution or ringer lactatesolution. The concentration of the compound in such solutions may be inthe range 1-60 g/liter.

In certain embodiments, exemplary compounds and processes for makingcompounds of the invention include the following:

The compounds may be in crystalline form, e.g., after crystallizationand/or recrystallization in an alcohol, ketone, ester, or combinationthereof. For example, the compounds of the present invention may becrystallized and/or recrystallized from an alcohol such as ethanol.

Exemplary compounds of the invention include the following:

It has been found that when used in the form of nanoparticles, thecompounds of the claimed invention achieve higher blood levels. In oneembodiment, the present invention includes compound 2250 alone or incombination with taurolidine and/or taurultam. For example, the presentinvention includes nanoparticles of the compounds of the presentinvention encapsulated in capsules.

In certain embodiments, the invention also relates to derivatives of theabove compounds having, e.g., activity as described herein of saidcompounds, for example, at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 100%, or more, of said activity.

In certain embodiments, the invention also relates to compositionscontaining the compounds described herein, including pharmaceuticallyacceptable solutions of said compounds, as well as orally administrablecompositions such as capsules and tablets containing said compositions.

In certain embodiments, the compounds of the present invention can beadministered to a subject or patient by any suitable means, for example,in solution, e.g., locally, systemically such as by intravenousinfusion, or the like.

Synthesis of 2250

-   -   sublimes in a vacuum at ˜70-80° C.

-   Starting materials:

-   Isethionic Acid,

-   Carbylsulfat, Taurin, Taurinamide,

-   Cysteine, Isethionic Acid, inter alia

-   Synthesis 1

I.

Chemical Synthesis

-   -   ethylenoxide with bisulfite

II. Isethionic Amide

Possible Alternative Chemical Synthesis Steps for 2250

Several Alternative Synthesis Steps for 2250 and 2255

I. Starting materials 2250/2255

-   -   Synthesis sodiumisethionate from        Ethylenoxide+Sodiumhydrogensulfite        II. Reaction of Amine with Carbylsulfate

III. Exemplary Synthetic Protocols

I. Synthesis of 2244

-   2.15 g of pure 1907 was dissolved in 100 ml acetic acid ethyl ester,    and catalyzed using 0.5 g palladium on activated carbon. The    solution was hydrogenated at room temperature and atmospheric    pressure. The hydrogenation was complete after about 15 hours and    the absorbed amount of hydrogen was 450 ml.-   The hydrogenation was evacuated 3 times, each time with nitrogen,    and then the reaction mixture was filtered through a filter aid    (diatomaceous earth). The clear colorless ethyl acetate solution was    concentrated and dried in a rotary evaporator.-   Yield: 1.25 g, which was innoculated with crystalized 2244.-   Melting point: 42-44° C.-   IR: corresponds to 2244, 99.3% pure.

II. Synthesis of 2244

-   5 g (0.023 mol) of 2264/1907 was boiled in 50 ml of concentrated HCl    for 3 hours under reflux, then allowed to cool to room temperature    and separated with 30 ml of dichloromethane in a separating funnel.    The aqueous phase was evaporated in a rotary evaporator and dried. A    yellow oil remains which slowly crystallized after seeding with 2244    crystals.-   IR corresponds to the substance 2244.-   Recrystallized from ethyl acetate.-   0.7 g obtained (24%).-   Melting point: 44-45° C.-   IR corresponds to the reference substance.

III. Synthesis of 2244

wherein Ph is a phenyl group.

-   230 mg 2269 was dissolved in 2 ml NaOH (1 N) and refluxed at boiling    with a reflux condenser for 15 minutes. The clear solution was    cooled to 20° C. and acidified with hydrochloric acid. The resulting    precipitate was filtered off under vacuum and dried.-   Yield: 110 mg.-   Melting point: 114-116° C.-   IR showed 99% benzoic acid as by-product.-   The acidic solution was concentrated to dry it on a rotary    evaporator and the solid was boiled with acetic ester. The ethyl    acetate solution was filtered and concentrated to dryness under    vacuum.-   Weight: 110 mg. Oil was contaminated with oil and the IR peak for    2244 (isethionic acid amide) was unclean.-   The 110 mg was recrystallized from acetic ester.-   Yield: 65 mg, Melting Point: 43-45° C.-   IR corresponded to 52% 2244.

IV. Synthesis of 2244

wherein Ph is a phenyl group.

-   1.15 g 2269 was dissolved in 10 ml NaOH (1 N) and refluxed at    boiling for 15 minutes. The clear solution was cooled to 20° C. and    acidified with hydrochloric acid. The resulting precipitate was    filtered off under vacuum and dried.-   Yield: 0.5 mg.-   Melting point: 114-116° C.-   IR showed 82% benzoic acid by-product as control substance.    Hydrolysis is not complete.-   The acidic solution was concentrated to dry it on a rotary    evaporator and the solid was boiled with acetic ester. The ethyl    acetate solution was filtered and concentrated to dryness under    vacuum.-   Weight: 0.8 g. Oil was contaminated with oil and the IR peak for    2244 (isethionic acid amide) was unclean.-   The 0.8 g was recrystallized from acetic ester.-   Yield: 160 mg, Melting Point: 43-45° C.-   IR corresponded to 26% 2244.

V. Synthesis of 2244

-   215 g 0.1 Mol 2264 and 1000 ml of concentrated hydrochloric acid    (ca. 36%) were boiled together for 30 minutes under reflux. The 2264    resolved and there was an oily layer. The reaction mixture was    allowed to cool and transferred to a separatory funnel where the oil    was separated from the water phase. The acidic aqueous solution in    which should be solved isethionic acid amide (2244) was concentrated    at 50° C. in a rotary evaporator almost to dryness. The yellow oily    residue was placed overnight in the refrigerator and 32.3 g of clear    crystals were filtered off under vacuum. Mp 43-45° C. IR: in oxygen    having peaks at the following wave numbers 655.82, 729.12, 844.85,    898.86, 947.08, 1003.02, 1060.88, 1134.18, 1236.41, 1288.49,    1317.43, 1408.08, 1572.04, 3105.5, 3209.66, 3313.82, and 3427.62    cm⁻¹ as shown in FIG. 7. The mother liquor was concentrated to    complete dryness.

VI. Synthesis of 2244

-   21.5 g 0.1 Mol 2264 and 100 ml of concentrated hydrochloric acid    (ca. 36%) were boiled together for 30 minutes under reflux. An oily    layer formed and the reaction mixture was allowed to cool in a    separatory funnel where the oil was separated from the water phase.    The acidic aqueous solution in which the isethionic acid    amide (2244) was dissolved and shaken 2 times with methylene    chloride, the methylene chloride was separated, and the acidic water    solution was concentrated in a rotary evaporator at 50° C. to    dryness. The yellow oily residue was placed overnight in the    refrigerator and 12.3 g of oil was obtained. Mp.: 41-43° C. Analysis    of the product showed that corresponds 99.8% to 2244 by IR.-   Distillation Experiment:-   12.3 g were distilled under high vacuum:

Outside Inside Temperature Temperature Vacuum 190-210° C. 183-186° C.0.1 mm

-   Weight: 9.3 g of oil which was solid at room Temperature Mp: 43-45°    C.

VII. Synthesis of 2244

-   2.0 g of pure compound 1907 was dissolved in 200 ml acetic ester and    0.5 g palladium/activated carbon was added and the mixture was    autoclaved at 100° C. and hydrogenated at 50° C. After 6 hours    run-time, the reaction mixture was allowed to cool overnight, and    was then filtered and concentrated to dryness under vacuum.-   Wt.: 1.7 g oil—added CH2Cl2 and shaken, then allowed to stand—then    suction filtered result in crystalline solid having Wt.: 0.6 g,    melting point ca. 40° C.-   For analysis 0.2 g of two-times acetic ester was added to    crystallize. Melting point 43-44° C.

VIII. Synthesis of 2244

-   2.15 g of pure 1907 was dissolved in 100 ml acetic acid-ethyl ester,    then added to 0.5 g palladium/activated carbon. Then the mixture was    hydrogenated at room temperature and atmospheric pressure.    Hydrogenation was terminated after approximately 15 hours. The    absorbed amount of hydrogen was approximately 450 ml. The hydrogen    was then evacuated 3 times and flushed with nitrogen, and then each    reaction mixture was filtered through diatomaceous earth (celite).    The clear, colorless solution, ethyl acetate was evaporated to    dryness on a rotary evaporator.-   Wt.: 1.25 g oil which crystallized after seeding with 2244 crystals.-   Melting point: 42-44° C.-   IR: corresponds to 99.3% 2244.

IX. Synthesis of 2250

-   1.2 g pure 2245 pure was dissolved in 150 ml acetic acid purely    solved at 60° C. 0.3 g of palladium on activated carbon was added    and was stirred at 75° C. and the mixture was hydrogenated at    atmospheric pressure.-   Hydrogenation was stopped after 7 days. The absorbed amount of    hydrogen was approximately 480 ml.-   The hydrogen was evacuated and purged 3 times with nitrogen.-   Then the reaction mixture was filtered at 70° C. through a filter    aid (Diatomaceous earth).

The clear warm glacial acetic acid solution was cooled down to roomtemperature and white crystals were suction filtered.

-   Weight: 0.74 g, Melting Point: 225-227° C.-   IR: 2245 corresponds to the starting material-   The mother liquor was concentrated on a rotary evaporator to    dryness.-   Weight: 0.38 g of impure material was extracted with ethyl acetate.-   The solution was concentrated.-   Ethyl acetate Soluble Portion: Semi-solid substance obtained by    sublimation;-   Obtained 0.15 g semi-solid substance that was recrystallized from a    few drops of water-   Yield: 70 mg, Melting Point: 95-98° C.-   IR corresponded to 98% 2250.    X. 1-step Synthesis in High Yield of Sodium 2-benzylether    ethanesulfonate

-   10.5 g sodium 2-bromoethanesulfonate was added to a solution of 110    ml benzyl alcohol and 1.15 g sodium benzyloxide.

Then the mixture was boiled under reflux four times. The mixture wasthen concentrated under vacuum to dryness and then boiled with ethylalcohol three times. The alcohol was filtered and concentrated todryness.

The yield was 9.8 g and was confirmed by UV and IR.

Pure crystals were obtained by boiling the resultant sodium2-benzylether ethanesulfonate in ethyl alcohol, filtering, then coolingthe solution to crystallize pure sodium 2-benzylether ethanesulfonatecrystals out of solution.

XI. Synthesis of 2250

-   6.3 g vinylsulfonamide (from 2258),-   50 ml of concentrated formic acid, and-   1.1 g of paraformaldehyde were combined for 2 hours at reflux to    produce compound-   2250. Then, the clear acidic solution was concentrated on a rotary    evaporator to dryness.-   Residue is: 5.9 g of pale yellow, honey-like syrup.-   IR: Mixture of vinylsulfonamide and 2250-   A 2 grams was sublimated and a few crystals were obtained.-   Sublimate semisolid: IR: corresponds to 98% 2250.

XII. Synthesis of Vinylsulfonamide

-   Formyl isethionic chloride was placed in 50 ml of chloroform and was    placed in a 350 ml sulfonation flask and cooled to −10° C. Then 25%    ammonia gas was introduced. After introduction of the ammonia gas,    the weight of the chloroform/NH3 was found to be 5 g.-   From −3° C. to 2° C., the mixture was stirred slowly.-   To 9.0 g distilled 2249-   20 ml of chloroform was added drop wise. NH₄Cl precipitated    immediately.-   Then the ammonium chloride was filtered off under vacuum and the    clear chloroform solution was concentrated in a rotary evaporator    until dry.-   Yield: 6.3 g of clear, thin oil.-   IR: corresponds to 96% CH2═CH—SO2—NH2 (vinylsulfonamide).

XIII. Synthesis of 2261

-   300 g (1.26 mol) 2260 was weighed into a 750 ml multi-necked flask    with KPG-stirrer.-   415 ml trichloroethylene+phosphorus oxychloride (Density corresponds    to about 1.47 in 10% POCl₃) and-   150 ml phosphorus oxychloride and 5.7 ml DMF was warmed to 105° C.    while stirring.-   The mixture was allowed to react for 5 hours.-   The solid was filtered by vacuum and the liquid was distilled under    water-pump vacuum. The filter cake was washed with ethyl acetate.    After distilling off of trichloroethylene and phosphorus    oxychloride, the wash-acetate was transferred into a flask and also    distilled.-   250 g (1.07 mol-85%) of a yellow liquid was collected. IR    corresponds to 2261.

XIV. Synthesis of 2250 and 2255:

XV. New Synthesis schemes for compound 2250 and related compounds:

-   Starting materials:-   3-Hydroxypropane-1-sulfonic acid

-   3-Hydroxy-propane-sulfonic acid-y-sultone (1,3-Propanesultone)

-   3-Hydroxy-propane-2-sulfonic acid

-   2-Hydroxy-propane-1-sulfonic acid

-   Compounds (Tetrahydro-oxathiazine-dioxide):

-   Chemical Intermediates-   Protecting group: Benzyl chloride

-   Protecting group: Benzyl chioroformate

XVI. Synthesis of Precursor Compounds

-   Synthesis:-   83.9 g vinylsulphonic acid sodium was added to a solution of 400 ml    benzylalcohol and 0.5 g sodium (catalytic amount) was added. The    mixture was warmed with stirring to 150° C. and most of the    vinylsulphonic acid sodium went into solution. After 3 hours, the    mixture was allowed to cool overnight and a thick solid    crystallized. This solid was vacuum-filtered and then suspended in    ethyl alcohol, vacuum-filtered and dried.-   Yield: 94.0 g, IR: corresponds to the desired compound (61.2% pure).

XVII. Synthesis of 1905

-   60 grams of vinylsulfonic acid sodium were added to a solution of    1000 ml benzylalcohol and 0.5 g of sodium. Then, the whole mixture    was stirred under reflux and heated. After approximately 3 hours,    the excess benzyl alcohol was distilled and removed by vacuum and    the rest was boiled with alcohol. The alcohol solution was filtered,    concentrated, crystallized to about ½,-   37.3 g of a yellow cotton-wool-like substance was obtained.-   The procedure was also repeated with 250 g vinylsulfonic acid sodium    and 2 liters of benzyl alcohol, processed as above and about 208 g    was crystallized.-   The procedure was also repeated with 100 g vinylsulfonic acid sodium    and 1 liter of benzyl alcohol, processed as above and about 105 g    was crystallized.

The procedure was also repeated with 200 g vinylsulfonic acid sodium,processed as above and about 130 g was crystallized.

XVIII. Synthesis of 1906

-   6.7 g of 1905 (recrystallized) was added to 50 ml thionyl chloride    and 1 ml dimethyl formamide. The sodium salt dissolved immediately    and the mixture was heated to 40-50° C., let stand overnight at    20° C. and vacuumed until concentrated. Yield: 9.8 g, which was    added to 50 ml NaOH 2N and stirred well. The NaOH solution was    washed with CHCl₃ and then shaken with concentrated HCl to    precipitate and captured with Na₂SO₄, then dried and distilled.-   The process was repeated with 208 g 1905 mixed with 1000 ml thionyl    chloride and 10 ml dimethyl formamide. The mixture was refluxed and    the excessive thionyl chloride was distilled off until dry. The    yield was 250 g, which was processed as above.

XIX. Synthesis of 1907

-   9.8 g of 1906 was dissolved in chloroform (CHCl3) (turbid) and    concentrated into a portion of 150 ml concentrated ammonia in water    and stirred. Stirring was continued for 3 hours with heating to    40-50° C. Then, the mixture was dried under vacuum and concentrated.-   Yield: 3.1g dark oil-   The 3.1g dark oil was added to 50 ml NaOH 2N and stirred well. The    NaOH solution was washed with CHCl₃ and then shaken with    concentrated HCl to precipitate and captured with Na₂SO₄, then dried    and distilled. Yield: 2.5 g oil-   For analysis, a sample of 0.5 g was condensed at a temperature of    160° C., became solid and crystallized 3 times from ethyl    acetate/benzene.-   Melting point: 75-76° C.-   Molecular formula: C₉H₁₃NO₃S-   MW: 215.2-   Calculated: C=50.23%, H=6.09%, N=6.51%, S=14.86%-   Actual: C=50.14%, H=6.15%, N=6.35%, S=14.79%

XX. Synthesis of 1908

-   1.2 g of 1907 was dissolved in 200 ml ethyl acetate and 0.4 g Pd    activated carbon was added. The mixture was hydrogenated in a    hydrogenated autoclave at 100 and at 50° C. for 4 hours. The mixture    was left under pressure for a weekend at room temperature.-   Then the ethyl acetate solution was filtered and dried under vacuum.-   Yield: 1.1 g oil.

XXI. Synthesis of 1908

-   2 grams of 1907 were dissolved in 200 ml ethyl actetate and 0.5 g-   Pd/Palladium/activated carbon was added. The mixture was    hydrogenated in a high pressure autoclave at 100 and at 50° C. After    6 hours, the reaction mixture was left to cool overnight, then    filtered and distilled under vacuum until it dried to a residual    oil.-   Yield: 1.7 g oil.-   CH2Cl2 was added, agitated and allowed to stand, crystallized, and    separated with suction under vacuum. Weight: 0.6 g, melting point    about 40° C.-   Analysis:-   0.2 g recrystallized 2 times from ethyl actetate.-   Melting point: 43-44° C.-   Molecular formula: C₂H₇NO₃S-   MW: 125-   Calculated: C=19.22%, H=5.65%, N=11.21%, S=25.65%-   Actual: C=19.20%, H=5.67%, N=11.07%, S=25.73%

XXII. Synthesis of 1909

19.9 grams of 1906 were dissolved in 100 ml chloroform and added into asolution of 23 grams pure benzylamine and 200 ml pure chloroform.Immediately, benzylamine hydrochloride precipitated and the reactionmixture became warm. The mixture was then refluxed and the hydrochloridecompound was separated by suction and the clear CHCl3-mother liquor wasput into vacuum for drying.

-   Yield: 27 g yellow clear oil that slowly became solid.-   The 27 g was dissolved into about 20 ml ethyl acetate and N-hexane    (q.s.) was added so that the solution became nearly turbid. The    mixture was set aside in the cold overnight and it crystallized.-   Yield: 9.2 g, melting point: 50-53° C.-   For analysis, 1 g in N-hexane was recrystallized three times.    Melting point 56-57° C.

XXIII. Synthesis of 2260

-   0.675 mol of isethionic acid sodium salt (100.0 g) and 2.02 mol    benzylchloride (233 mL) were mixed in a 750 mL multi-necked flask    with KPG-stirrer. The mixture was heated at 70° C. inside    temperature (95° C. outside temperature) and then-   Triethylamine (120 mL) was added drop wise over one hour and the    outside temperature was increased to 125° C. and maintained.    Subsequently, outside temperature increased to 140° C., and the    inside temperature rose to 130° C. A solid clustered at the stirrer,    but went back into suspension. Hydrochloric acid vapors evolved.

30 mL of triethylamine was added drop wise and then reacted for 1.5 morehours. A viscous yellowish suspension formed. The product was allowed tocool to 50° C. inside temperature, then 300 mL water was added andvigorously stirred for 20 minutes and the mixture was transferred to a2L separatory funnel. Then, the flask was rinsed out with 100 mL ofwater.

The combined aqueous phases were washed twice with 280 mLdichloromethane.

-   The aqueous phase was held at 40° C., while KCl was added to the    solution until saturated (about 130 g KCl). The mixture was filtered    through a fluted filter and stored overnight in a refrigerator.-   The remaining solid was extracted and dried, resulting in 30.85 g,    yield of 17.9%.-   IR: OH band is present, similar to the precursor.-   The mother liquor was again treated with KCl and stored (at 35-40°    C.) overnight in the refrigerator.-   Solid from the second precipitation with KCl was filtered off and    dried, resulting in 60.0 g=34.9% and the IR corresponds to the    desired product.-   Solid 1: Was boiled with 150 mL EtOH and filtered while hot.-   By repeated precipitating with KCl, boiling and crystallization, 32    g of the product were obtained for a yield of 19%.

XXIV. Synthesis of 2256

-   40 g taurinamide hydrochloride, 18 g Sodium nitrite and 300 ml of    distilled water were boiled together under reflux until no more gas    was created. The clear yellow solution was then cooled to 50° C.-   30 ml of 1N NaOH was added to 10.5 g of acetaldehyde. The clear    yellow solution was left over the weekend under vacuum to dry. The    result was a rust-red honey-like residue weighing 37.6 g, which was    extracted with ethyl alcohol. The alcohol solution was filtered and    concentrated on a rotary evaporator to dry. The resulting dense oil    residue was dissolved with ethyl acetate. The ethyl acetate solution    was filtered, and concentrated.-   This resulted in 30.7 g of dense oil, rust-like color. From the    dense oil, white crystals were isolated. The melting point is about    114-116° C.-   The IR spectrum confirmed that the resulting compound had the    structure of compound 2256:

In certain embodiments, a sublimation apparatus, comprised of laboratoryglassware known in the art, may be used in a technique of sublimation topurify compounds according to the invention. In certain embodiments, asublimation vessel is heated under vacuum and under reduced pressure.The compound volatizes and condenses as a purified compound on a cooledsurface, leaving non-volatile residue impurities behind. This cooledsurface often takes the form of a cold finger. After heating ceases andthe vacuum is released, the sublimed compound can be collected from thecooled surface.

In one embodiment, substituted derivatives compound 2250 may beprepared. Substituted derivatives of compound 2250 include:

Wherein R may be H or alkyl or aryl. In certain embodiments, R is a C₁to C₆ alkyl. In certain embodiments, R is methyl.

In certain embodiments, derivatives of compound 2250 are preparedaccording to the following reaction scheme:

In one embodiment, this disclosure includes a method of killing tumorstem cells by administering to a subject in need thereof a tumor stemcell killing effective amount of taurolidine, taurultam, or a mixturethereof. The tumor stem cell killing effective amount of taurolidineand/or taurultam is less than an amount of taurolidine and/or taurultamrequired for killing tumor cells.

In some embodiments, the taurolidine, taurultam, or a mixture thereof isadministered in a tumor stem cell killing composition at a concentrationof about 0.01 to about 500 μg/ml. In some embodiments, the taurolidine,taurultam, or a mixture thereof is administered in a tumor stem cellkilling composition at a concentration of about 0.1 to about 100 μg/ml.In some embodiments, the taurolidine, taurultam, or a mixture thereof isadministered in a tumor stem cell killing effective composition at aconcentration of about 10 to about 50 μg/ml. Taurolidine is effective atkilling tumor stem cells in tissue culture in vitro at 0.01 μg/ml.

In some embodiments, the taurolidine, taurultam, or a mixture thereof isadministered in a tumor stem cell killing composition at a concentrationof about 0.001 to about 2%. In some embodiments, the taurolidine,taurultam, or a mixture thereof is administered in a tumor stem cellkilling composition at a concentration of about 0.01 to about 1.5%. Insome embodiments, the taurolidine, taurultam, or a mixture thereof isadministered in a tumor stem cell killing composition at a concentrationof about 0.1% to about 1%.

In one embodiment, the taurolidine, taurultam, or a mixture thereof isadministered for tumor stem cell killing to a subject in need thereof ata total daily dose of from about 0.01 g to about 50 g, about 0.1 g toabout 30 g, about 0.5 g to about 10 g, or about 1 g to about 5 g.

Tumor stem cell killing effective dosage amounts of the taurolidine,taurultam, or a mixture thereof are dosage units within the range ofabout 0.01-500 mg/kg, preferably 1-100 mg/kg per day, and mostpreferably 5-50 mg/kg per day.

In another embodiment, this disclosure includes a method of killingtumor stem cells by administering to a subject in need thereof acompound selected from the following compounds:

wherein each R is independently H, alkyl, or aryl,

which may be used in combination with taurolidine and/or taurultam. Sucha technique provides a method for killing tumor stem cells using atleast two compounds having different half-lives, and thereby broadeningthe pharmacokinetic effects obtained thereby. In one embodiment,compound 2250 may be used in combination with taurolidine and/ortaurultam.

EXAMPLES Example 1

Anti-neoplastic activity of compound 2250

Introduction

Based on the recognition of taurolidine as a powerful anti-neoplasticagent, the analogue 2250 was synthesized by Geistlich Pharma.

Material and Methods

Chemicals: The compound 2250 and taurolidin 2% solution were provided byGeistlich Pharma A G, Wolhusen, assignee of the present invention.

Cell lines: The human glioma cell line LN-229 was used as describedpreviously (Rodak et al. 2005) as well as the human colon adenocarcinomacell line SW480.

Cytotoxicity assay: Dissociated LN-229 cells were seeded into 96-wellplates at a density of 10⁴ cells per well in 100 μl of culture medium.Approximately 24 h later, when the cells had reached 70-80% confluency,the medium was changed and treatment with compound # 2250 (4.0-1000μg/ml), taurolidine (4.0-1000 μg/ml) or standard medium was started.Triplicate cultures were prepared for each sample. After 24 h ofincubation at 25° C., the remaining adherent viable cells were stainedusing crystal violet as described (Rodack et al. 2005). Cell viabilitywas determined by measuring the absorbancy at 540 nm. The results areexpressed as killing rate given by the difference between 100% of cellsand percentage of cells surviving. EC₅₀ values correspond to theconcentration inducing 50% cell death.

Results

Positive control: After incubating the human glioblastoma cells (LN-229)for 24 h with taurolidine, a concentration—dependent cytotoxicity wasdetermined (Tab. 1, FIG. 1) with an EC₅₀=45 μg/ml, a value whichcorresponds to earlier results obtained with this cell line (Rodack etal. 2005).

Test of 2250: When 2250 was incubated under the same experimentalconditions as taurolidine, a similar concentration-dependent loss ofcell viability was observed. The half-maximal concentration of inducingcell death was EC₅₀=50 μg/ul (Tab. 1, FIG. 1).

The results for SW480 cell cytotoxicities are shown in FIG. 2.

Discussion

The compound 2250 represents a new avenue in the search for novelantineoplastic agents of the taurolidine-type. Biologically, thecompound is as potent as taurolidine. Chemically, the compound showsstrikingly different features from taurolidine. By replacing a NH groupby an ether-oxygen, the double ring structure of taurolidine is avoided.Compound 2250 is a single ring structure and a close structural analogueof taurultam.

Mechanistically, the results show that the antineoplastic activity oftaurolidine is unlikely to be due to the formation of amethoxy-derivative, since 2250 is devoid of a methoxy group. Thecompound causes blebbing of tumor cells.

Summary

The compound 2250 shows potent antineoplastic activity in vitro, asdetermined for human glioblastoma cells (cell line LN-229). Its potency(EC₅₀=45 μg/ml) is comparable to that of taurolidine (EC₅₀=50 μg/ml) astested in the same cell line.

TABLE 1 Cytotoxicity of 2250 and taurolidine against LL-229 glioblastomacells. Concentration μg/ml 1000 500 250 125 62.5 31 15.5 8 4 —Taurolidine 0.109 0.098 0.165 0.305 0.317 1.132 1.434 1.478 1.530 1.435OD ± ± ± ± ± ± ± ± ± ± ± SD 0.010 0.007 0.002 0.008 0.008 0.042 0.0310.040 0.026 0.009 Comp. 2250 0.189 0.141 0.120 0.199 0.372 1.482 1.4821.527 1.477 1.483 OD ± ± ± ± ± ± ± ± ± ± ± SD 0.007 0.007 0.012 0.0140.006 0.099 0.029 0.033 0.069 0.013The values were measured in triplicate and the OD is the absorbance at540 nm plus minus standard deviation (SD). High values correspond tohigh cell viability.

Example 2

The new compound 2250 (Tetrahydro1,4,5-oxathizain-4-dioxid) was testedand found to have a very high level of antibacterial activity againstStaphylococcus aureus and Escherichia coli. The antibacterial activityagainst Staph. aureus is about double as high as Taurultam.

Example 3

In punch plate tests, Compound 2250 was tested and found highly activeagainst MRSA lines 188, 189, 193, 194 and 195.

By displaying a combination of antimicrobial and antineoplasticactivity, compound 2250 is particularly suitable for surgical oncology.

Example 4

Each of compounds identified herein as compound 2250, 2255, 2245, A1,A3, B1, B2, or B3 is tested against cancer cell lines of cancersidentified herein, and found to be active against such cell lines.

Example 5

Each of compounds identified herein as compound 2250, 2255, 2245, A1,A3, B1, B2, or B3is administered to patients having cancers identifiedherein, and is found to be effective in treating such cancers and safefor use in patients. Each of these compounds is administered withVitamin D3, a derivative, metabolite or analog thereof and thecombination is found to increase the anti-tumor effects of thecompounds.

Example 6

The half-life of compound 2250 in human fresh blood was measured at 37°C. in vitro by GC, PYE Unicam Series 204 FID.

-   Baseline Value: 49.0 ppm-   After 1 hour: 50.6 ppm-   After 2 hours: 47.6 ppm-   After 20 hours: 38.6-39.0 ppm.

Thus, the half-life of compound 2250 is greater than 24 hours in humanblood, which is significantly higher than the half-life of taurolidine,which was found to be ˜30 minutes using the same test.

Example 7

Tissue samples from high grade gliomas WHO grade IV from newly diagnosedpatients (medium age of 54±10 years) were minced mechanically, digestedenzymatically and the dissociated cells were filtered. The isolatedtumor cells were cultured as bulk cells. Cancer Stem Cells (CSCs) wereisolated by the formation of neurospheres under neurosphere conditions(using neurobasal medium) from the murine SMA 560 glioma cell line orfrom freshly isolated human glioblastoma cells.

Cytotoxicity Assay

Bulk glioma tumor cells were cultured and incubated with taurolidine ortaurultam for 24 h or 48 h as described previously (Rodak et al., J.Neurosurg. 102, 1055-1068, 2005). CSCs were cultured for 7 days andsubsequently exposed to taurolidine, taurultam or temozolamide for 24hours. The number of remaining adherent cells were stained (crystalviolet or Alamar Blue) and quantified by absorbance measurements (540nm). Cell survival was expressed as the percentage of cells survivingrelative to the number of cells surviving in untreated control cultures.The results are given as % killing rate or EC₅₀ as the dose required forhalf-maximal cytotoxicity.Results

Cytotoxicity of Taurolidine and Taurultam against Cancer Cells andCancer Stem Cells from the Mouse

The mouse SMA560 glioma cell line was used to provide tumor bulk cellsand CSCs. Following incubation of SMA560 bulk cells with variousconcentrations of taurolidine and taurultam (6.25, 12.5, 25, 50, 100,200 μg/ml), cytotoxicity was determined after 24 h and 48 h ofincubation. For both taurolidine and taurultam, a clear dose-dependentcytotoxicity was found with no major difference in potency between the24 h and 48 h time of incubation (FIG. 3A,B). The EC₅₀ value was 34.6pg/ml for taurolidine and 19.3 μg/ml for taurultam (FIG. 3C). Mouse CSCswere generated from the SMA560 glioma cell line and cultured for 7 days.The CSCs were treated with the same concentration of taurolidine andtaurultam as above and cytotoxicity was determined after 24 hours. Asshown in FIG. 4, both taurolidine and taurultam showed a dose⁻dependentcytotoxicity with an EC₅₀ of 12.5 μg/ml for taurolidine and EC₅₀ of 10μg/ml for taurultam against murine CSCs. These values demonstrate forthe first time that taurolidine and taurultam are effective against aCSC.

Taurolidine and Taurultam Induce Cell Death in Human CSC Isolated fromFour Different Glioblastoma Patients

CSCs were isolated from glioblastoma tissue resected from four patients.The same range of concentrations of taurolidine and taurultam wasapplied as above and the cytotoxicity was measured after 24 hours ofincubation with drug. All four glioblastoma CSCs tested (GBM #3, #4, #5and #6) were similarly sensitive to taurolidine and taurultam (FIG.5A,B). The mean EC₅₀ value of taurolidine was 13±2 μg/ml, the EC₅₀ valueof taurultam was 11±1.4 μg/ml (Table 2). In these experiments, thecytotoxic capacity of taurolidine and taurultam was compared with thatof temozolamide (TIM) applied in the concentration range of 5 μM to1,000 μM (FIG. 2C). The mean EC₅₀ value of TMZ was 68.5±26 μg/ml (Table2). Interestingly, this concentration is much higher than peak plasmalevels of TMZ measured in patients (13.7 μg/ml) (Portnow et al., ClinCancer Res 15, 7092-7098, 2009).

The results demonstrate that both taurolidine and taurultam areeffective against CSCs and this finding was established for glioma CSCsfrom two species, mouse and man.

The mouse CSCs were generated from a mouse glioma cell line (SMA 560).Remarkably, based on the EC₅₀ values, the CSCs were even more sensitiveto taurolidine and taurultam than the corresponding glioma bulk cells(about 3 fold for taurolidine and 2 fold for taurultam) (FIGS. 3, 4).

Human CSCs, freshly isolated from four human glioblastoma patients, werelikewise highly chemosensitive to both taurolidine and taurultam. TheEC₅₀ values for cytotoxicity were 13±2 ug/ml and 11±1.4 μg/ml,respectively (Table 2). These values demonstrate that the human CSCs,like their murine counterparts, are more sensitive to taurolidine andtaurultam (about 3 to 4 fold) than the human glioblastoma bulk cellswhich display EC₅₀ values in the range of 50 μg/ml (Rodak et al., J.Neurosurg., 102, 1055-68, 2005).

TABLE 2 Cytotoxicity Induced by taurolidine (Tau), taurultam (TT) ortemozolamide (TMZ) in cancer stem cells (CSC) derived from fourglioblastoma patients. EC₅₀ (μg/ml) = drug concentration resulting in50% cell death compared to untreated control cultures in vitro. CancerCytotoxicity Stem EC₅₀ (μg/ml) 24 h Cells n Taurolidine TaurultamTemozolamide GBM #3 3 15 10.5 84.4 (435 μM) GBM #4 2 12.5 12.5 97 (500μM) GBM #5 2 14 11 48.5 (250 μM) GBM #6 3 10 9 44 (230 μM) Mean ± SD 13± 2 11 ± 1.4 68.5 ± 26

Example 8

Taurolidine and taurultam were tested against cancer stem cells derivedfrom a murine glioma cell line and human cancer stem cells. Taurolidineand taurultam were found to exert potent anti-neoplastic activityagainst cancer stem cells derived from a murine glioma cell line(EC₅₀=12.5 μg/ml for taurolidine, EC₅₀=10 μg/ml for taurultam) as wellas against human cancer stem cells, freshly isolated from fourglioblastoma patients (EC₅₀=13±2 μg/ml for taurolidine; EC₅₀=11±1.4μg/ml for taurultam).

Example 9 Antineoplastic Effect on Pancreatic Stem Cell-LikeMulticellular Spheroid Cultures.

Multicellular spheroids are composed of tumor cells growing in a3-dimensional structure stimulating the growth, micro-environmentalconditions and stem cell-like characteristics of real tumors. Themulticellular tumor spheroid (MCTS) model compensates for many of thedeficiencies seen in monolayer cultures. Spheroids on the scale of200-500 μm develop chemical gradients of oxygen, nutrients, andcatabolites, while having morphological and functional features similarto tumors. Therefore, assays utilizing the MCTS model allow for theassessment of drug penetration and are more predictive of in vivosuccess compared with monolayer cultures. MCTS assays are a tumor modelsystem of intermediate complexity between standard monolayer and tumorsin vivo.

Pancreatic tumor cells (Panc Tu-1, BxPC-3, Mia Paca-2, ASPC1) andpancreatic primary tumor cells (Bo80) were seeded in ultra-low adhesionplates in special stem cell media.

Pancreatic tumor cells (ASPC1, Mia Paca-2, Panc Tul, BxPC-3) andpancreatic primary tumor cells (Bo80) were raised in monolayer culturebefore seeding in ultra low adhesion plates under conditions of specialstem cell media to form multicellular spheroids and passed through acell strainer to exclude aggregates.

Half-maximal inhibition of cell viability was achieved with 750-1000 μMof compound 2250 in tumor cell lines AsPC-1, BxPC-3 and HCT-116. Theseeffects are similar to those observed in glioma cell line LN-229. Theinduction of cell death was due to apoptosis and necrosis (most likelynecroptosis). It was found that the induction of this programmed celldeath was prevented by addition of the reducing agent N-acetylcysteineand that caspases are not involved. Thus, there is a redox-directedmechanism of action.

The growth of pancreas tumor cells (AsPC-1, BxPC-3 and HCT-116) wasinhibited by compound 2250 with a half-maximal concentration of 300 μM,which is considerably lower than the concentration needed to elicitcytotoxicity.

As shown in FIG. 8, multicellular pancreatic tumor (Panc Tul or BxPC-3)spheroids were tested as control, taurolidine-treated (500 μM) orcompound 2250-treated (1000 μM) samples for 48 hours (columns labeledA). After treatment, each of the whole cell suspensions was passedthrough a 45 μm cell strainer again to analyze residual aggregates fortheir stability (columns labeled B).

FIGS. 9A and 9B show the results of FACS analysis of the Panc Tulmulticellular spheroid cultures CD133 content. CD133 is a known andwell-established hallmark of stem cells. The results show that theamount of CD133-positive cells in multicellular spheroid cultures ofPanc Tul was enriched 10-fold compared to Panc Tul grown in monolayerculture (B). Isotype IgG was used as negative control (A). The resultsdemonstrate that taurolidine and compound 2250 have an antineoplasticeffect on the pancreatic stem cell like multicellular spheroid cultures.

Example 10

In vivo study of taurolidine and compound 2250 as antineoplastic agentsin malignant pancreatic carcinoma.

The effects of taurolidine and compound 2250 were analyzed on nude mice(NMRI-Foxn1 nu/nu). 1×10⁷ tumor cells (PancTu-I and MiaPaca 2) wereinjected subcutaneously into the flank. The animals were randomized intothree groups: the control group; the group treated i.p. with taurolidine(TRD), and the group treated i.p. with compound 2250 (NDTRLT).

Tumors were grown to a size of 200 mm³ before the treatment was started.Mice were treated on alternating days with 500 mg/kg*body weight (BW).

As shown in FIG. 10A, administration of taurolidine decreased MiaPaca2tumor volume significantly compared to control (by about 2-fold).

As shown in FIG. 10B, administration of compound 2250 decreased MiaPaca2tumor volume significantly compared to control (by over 3-fold).

As shown in FIG. 10C, administration of taurolidine decreased PancTu Itumor volume significantly compared to control (by about 3-fold).

As shown in FIG. 10D, administration of compound 2250 decreased PancTu Itumor volume significantly compared to control (by about 2-fold).

The applied taurolidine and compound 2250 dosages showed no toxic effecton the mice during the study. In both tumor cell line models, asignificant reduction of tumor growth was obtained.

Tumor growth (volume) was significantly reduced from day 9 onwards(PancTul) and day 11 onwards (MiaPaca2) versus controls. The dose of 500mg/kg i.p. was well tolerated with no overt sign of toxicity.

As shown in FIG. 11A, a xenograft model of pancreatic primary tumors (Bo73) was observed for 15 days and it was found that administration oftaurolidine slightly reduced relative tumor volume compared to controland administration of compound 2250 further reduced relative tumorvolume compared to control. However, the differences in tumor volumeswere not statistically relevant, likely due to the short duration of thestudy and the slow growth rate of the tumors. In FIG. 11B, a xenograftmodel of pancreatic primary tumors (Bo 70) was observed for 23 days andit was observed that administration of taurolidine and compound 2250significantly reduce tumor volume compared to control.

Administration, e.g., intraperitoneally, of taurolidine and/or compound2250 inhibits tumor growth in vivo.

What is claimed is: 1-51. (canceled)
 52. A method for producing compound2260 comprising the following reaction:


53. A process for producing

(sodium 2-benzyletherethane sulfonate) comprising the followingreaction:


54. The process of claim 53, wherein the process comprises adding sodium2-bromoethanesulfonate to a solution of benzyl alcohol and sodiumbenzyloxide to form a mixture, boiling the mixture to reflux four times,concentrating under vacuum until dry, boiling with ethyl alcohol,filtering the ethyl alcohol, and concentrating to dryness to obtainsolid sodium 2-benzyletherethanesulfonate.
 55. The process of claim 54,further comprising boiling said solid sodium 2-benzyletherethanesulfonate in ethyl alcohol, filtering, then cooling to obtainsodium 2-benzylether ethanesulfonate crystals having increased purity.56. A process for producing a compound having the formulaPh—CH₂—O—CH₂—CH₂—SO₃Na comprising the following reaction:


57. The process of claim 56, which comprises the following stepssequentially: adding vinylsulphonic acid sodium to a solution of benzylalcohol and a catalytic amount of sodium to form a mixture, warming themixture with stirring to dissolve most or all of the vinylsulphonic acidsodium, cooling the warmed mixture to obtain crystals, vacuum filteringthe crystals, suspending the filtered crystals in ethyl alcohol, vacuumfiltering the suspended crystals, andPh—CH₂—O—CH₂—CH₂—SO₃Na drying the vacuum filtered crystals to obtain 58.A method for producing

comprising the following reaction:


59. The process of claim 58, which comprises the following stepssequentially: adding vinylsulphonic acid sodium to a solution of benzylalcohol and a trace amount of sodium, stirring the mixture under reflux,distilling off the benzyl alcohol under vacuum, boiling the remainderwith alcohol, filtering the alcohol, concentrating the alcohol andcrystallizing the resulting reaction product to obtain


60. A process for producing a

salt comprising the following reaction:

salt.
 61. The process of claim 60, comprising the following stepssequentially: mixing isethionic acid sodium salt with benzyl chloride,heating the mixture, adding triethylamine dropwise to the mixture,cooling the mixture, adding water to the mixture and stirring to obtainan aqueous phase and an organic phase, separating the aqueous phase fromthe organic phase, washing the aqueous phase with dichloromethane,adding KCl to the aqueous phase, filtering, refrigerating the aqueousphase, extracting remaining solid, treating the aqueous phase with KCl,refrigerating the aqueous phase, filtering off solid product from theaqueous phase and drying the solid to obtain


62. A method for producing a

salt comprising the following reaction:

salt, wherein X is a leaving group.
 63. A process for producing a

salt comprising the following reaction:

salt, wherein X is a leaving group and each R is, independently, analkali metal.
 64. The process of claim 63, wherein the process comprisesadding a 2-haloethanesulfonate salt to a solution of benzyl alcohol andan alkali metal salt of benzyloxide to form a mixture, boiling themixture to reflux, concentrating under vacuum until dry, boiling with analcohol, filtering the alcohol, and concentrating to obtain solid2-benzyletherethanesulfonate salt.
 65. The process of claim 64, furthercomprising boiling said solid 2-benzylether ethanesulfonate salt inalcohol, filtering, then cooling to obtain 2-benzylether ethanesulfonatesalt crystals having increased purity.
 66. A process for producing acompound having the formulaPh—CH₂—O—CH₂—CH₂—So₃ ⁻R⁺, comprising the following reaction:

wherein each R is independently an alkali metal.
 67. The process ofclaim 66, which comprises the following steps sequentially: addingvinylsulphonic acid salt to a solution of benzyl alcohol and a catalyticamount of an alkali metal to form a mixture, warming the mixture withstirring to dissolve most or all of the vinylsulphonic acid salt,cooling the warmed mixture to obtain crystals, vacuum filtering thecrystals, suspending the filtered crystals in alcohol, vacuum filteringthe suspended crystals, and dryingPh—CH₂—O—CH₂—CH₂—SO₃ ⁻R⁺ the vacuum filtered crystals to obtain
 68. Amethod for producing

comprising the following reaction:

wherein each R is independently an alkali metal.
 69. The process ofclaim 68, which comprises the following steps sequentially: addingvinylsulphonic acid salt to a solution of benzyl alcohol and a traceamount of an alkali metal, stirring the mixture under reflux, distillingoff the benzyl alcohol under vacuum, boiling the remainder with alcohol,filtering the alcohol, concentrating the alcohol and crystallizing theresulting reaction product to obtain


70. A process for producing

comprising the following reaction:

wherein X is a leaving group and wherein R is an alkali metal.
 71. Theprocess of claim 70, comprising the following steps sequentially: mixingisethionic acid salt with benzyl halide, heating the mixture, adding anorganic base to the mixture, cooling the mixture, adding water to themixture and stirring to obtain an aqueous phase and an organic phase,separating the aqueous phase from the organic phase, washing the aqueousphase with dichloromethane, adding a first inorganic salt to the aqueousphase, filtering, refrigerating the aqueous phase, extracting remainingsolid, treating the aqueous phase with the first inorganic salt or asecond inorganic salt, refrigerating the aqueous phase, filtering offsolid product from the aqueous phase and drying the solid to obtain